Nitric oxide (NO) is a free radical involved in the regulation of several functions of the male genitourinary system. It is produced by neurons and the endothelium and epithelia of reproductive system; it mediates penile erection and regulates sperm motility, viability, and metabolism. Here we show that human spermatozoa exhibit a detectable NO synthase (NOS) activity, measured both as ability of the intact sperm and cell lysate to convert L-[3H]arginine into L-[3H]citrulline and as 24 h accumulation of extracellular nitrite in intact sperm suspensions. NOS activity (identified as an endothelial isoform) was inhibited by L-canavanine and N(G)-monomethyl-L-arginine, and nitrite accumulation was inhibited by the NO scavenger hemoglobin; both enzyme activity and nitrite production were increased by a 24 h incubation of spermatozoa with protein-enriched extracts of human follicular fluid (PFF); a significant increase of citrulline synthesis was observed only after a 4 h incubation with 40% PFF, a time period during which acrosomal reactivity was significantly increased. PFF-induced acrosomal reaction was inhibited by L-canavanine and hemoglobin, and the NO donors sodium nitroprusside (SNP), S-nitroso-N-acetyl-penicillamine (SNAP), and DETA NONOate were able to increase the percentage of reacted spermatozoa. Our results suggest that NO synthesized by human sperm may play a role in follicular fluid-induced acrosomal reaction.